This invention relates to novel functionallized liposomes used in the medical fields such as clinical examinations, diagnosis, remedy, etc. and a process for production thereof.
More particularly, this invention relates to novel functionallized liposomes comprising a high-molecular-weight amphiphilic compound as one of matrix material, and a process for production thereof. The functionallized liposomes of this invention have a very high encapsulation efficiency, and make it possible to immobilize immunological substances, biological active substances, etc. on liposomes efficiently with a sufficient binding rate without injuring the liposome.
Since Bangham et al. found that when phospholipids which are surfactants derived from biomembranes were suspended in water, closed vesicles composed of lipid bilayers were formed (J. Mol. Biol. 13,238 (1965)), these vesicles have been called liposomes and in recent years, their preparation method and application in various fields have energetically been investigated. In addition, it has recently also been found that besides phospholipids, highly molecular-designed artificial synthetic lipids (amphiphilic substances having molecular weight of about 1,000) form vesicles like liposomes (J. H. Fendler, "Membrane Mimetic Chemistry", John Wiley & Sons, N.Y., (1982)). Liposomes are noted very widely in the fields of clinical examinations, diagnosis, medicines, etc. because of their biocompatibility, not only as mere models of functions and structural characteristics of cells but also as artificial erythrocytes, a means for introducing genetic information into protoplasts in genetic engineering, carriers for immobilizing enzymes, and drug delivery systems for developing and improving remedies for cancer and other incurable diseases.
As conventional methods for preparing encapsulating liposomes, there have been reported many methods such as the most fundamental method comprising formation of a lipid thin film, followed by treatment by vortexing (A. D. Bangham et al, J. Mol. Biol., 13, 238 (1965)), sonication or the like (C. Hudng et al, Biochemistry, 8, 344 (1969)), the reverse-phase evaporation method (REV method) using organic solvents (F. Szoka et al, Proc. Natl. Acad. Sci., U.S.A., 75, 4194 (1978)), the ethanol infusion method (S. Batzri et al, Biochem. Biophs. Acta., 298, 1015 (1973)), the ether infusion method (D. Deamer et al, Biochim. Biophys. Acta., 443, 629 (1976)), and methods using surfactants (J. R. Slack et al, Biochim. Biophys. Acta., 323, 547 (1973)). Various types of liposomes can be prepared by these preparation methods. Liposomes range in size widely from 0.02 to 10 .mu.m diameter, and according to their size and structure, they are usually roughly divided into three types, namely, multilamellar vesicles (abbreviated as MLVs, 0.3 to 10 .mu.m in diameter), small unilamellar vesicles (abbreviated as SUVs, 0.025 to 0.1 .mu.m in diameter), and large unilamellar vesicles (abbreviated as LUV, 0.2 to 2.0 .mu.m in diameter) (F. Szoka et al, Ann. Rev. Biophys. Bioeng., 9, 467 (1980)). In addition to them, there are oligolamellar vesicles, and liposomes obtained particularly by the REV method are separately dealt with as reverse-phase evaporation vesicles (abbreviated as REVs) in some cases. The encapsulation efficiency indicated the rate of retention of encapsulated substances in liposomes, and is defined as the volume of water held per mole of lipid. This value varies depending on the kind of liposome, and now it is considered that liposomes prepared by the REV method have the highest encapsulation efficiency and that LUV is the second to be able to encapsulate proteins and nucleic acids. However, any of these liposomes obtained by conventional preparation methods are not always sufficient in captured volume for encapsulating high-molecular-weight substances such as enzyme proteins, and the advent of liposomes having a larger captured volume and a higher encapsulation efficiency has been eagerly waited for in consideration of application in the field of medical treatment, diagnosis, clinical examinations, etc., for example, encapsulation of expensive drugs in liposomes and preparation of highly sensitive diagnostic pharmaceutical compositions.
On the other hand, when used for clinical examinations or as diagnostic drugs or drug delivery systems for curative drugs for cancer and other incurable diseases, liposomes are used usually after immobilization thereon of antigen, antibody, other proteins, etc.
As methods for immobilizing antigen, antibody, other proteins, etc. on liposomes, there have so far been known the following methods (i) to (iv).
(i) A method which comprises introducing a hydrophobic group into a protein to be immobilized on liposomes, thereby imparting thereto affinity for liposomes, and then incorporating the thus treated protein into separately prepared liposomes (Biochemical et Biophysical Acta., 812, 116 (1985), etc.).
(ii) A method which comprises mixing a substance having a chemically modificable group, e.g., ganglioside (molecular weight: about 2,000) which is a low-molecular-weight glycolipid, previously at the time of liposome preparation, oxidizing its saccharide moiety with an oxidizing agent to form an aldehyde group, reacting the aldehyde group with the amino group of protein to form a Schiff base, and thereby combining liposome with the protein (Biochemical et Biophysical Acta, 640, 66 (1981), etc.).
(iii) A method which comprises mixing a lipid having a functional group reactive to SH group at the time of liposome preparation as in (ii) above, and reacting with protein separately modified with a sulfhydrylating agent to incorporate the same (Journal of Immunological Method, 75, 351 (1984); Biochemical and Biophysical Research Communications, 117, 399 (1983); Japanese Patent Application Kokai (Laid-Open) No. 117159/85, etc.).
(iv) A method which comprises binding the respective functional groups of liposomes and protein to each other by use of a cross-linking agent, a condensing agent or the like without any previous treatment of the liposomes and the proteins (Biochemical and Biophysical Research Communications, 89, 1114 (1979); Liposome Technology, 155 (1983), CRC Press, etc.).
However, in all these methods protein such as antigen or antibody is immobilized by reacting near the surface of liposomes, and therefore the membrane structure is very liable to be injured at the time of reaction and the binding rate of protein is not always sufficient, resulting in low efficiency, because of the steric hindrance of liposome matrix.
In general, when liposomes on which antibody or antigen is immobilized are used, for example, for clinical examinations or in diagnostic drugs, liposomes which readily undergo immunolysis by the action of complement, etc. after antigen-antibody reaction are preferred and hence the development of liposomes which are susceptible to immunolysis is desired.